The lab continues to use cell culture and mouse models to study the function of PKD1, PKD2 and PKHD1, the genes responsible for the most common forms of autosomal dominant and autosomal recessive polycystic kidney disease. As we reported last year, we have performed transcriptional profiling of a large set of male and female kidneys of adult mice induced at P40 and followed for up to 200+ days, along with metabolomics and lipidomics analyses of a subset of male kidneys. We found that females have a significantly less severe kidney phenotype and correlated this protection with differences in lipid metabolism. We showed that sex is a major determinant of the transcriptional profile of mouse kidneys and that some of this difference is due to genes involved in lipid metabolism. Pkd1 mutant mice have transcriptional profiles consistent with changes in lipid metabolism and distinct metabolite and complex lipid profiles in kidneys. We next hypothesized that if lipid metabolism plays a role in cystogenesis, then manipulating the lipid content of diets could potentially alter disease progression. Since breast milk fatty acid composition can be manipulated through diet, we tested the hypothesis by feeding nursing dams two chows that differ mainly in their lipid composition to nursing moms and young pups. We initially used a tamoxifen-inducible line (ER-Cre), induced at 7 days of age (P7) and harvested at P21, but we have since repeated the study using a second mouse line that expresses the cre-recombinase under the control of Ksp and resulting in epithelial deletion of Pkd1 in mid-gestation. In this latter study, we harvested the pups at P14. In both lines, the change in diet had no significant effect on body weight, and on both diets kidneys were quite cystic. However, in both lines, decreasing the lipid content of the diet by what seemed a trivial amount (from 7.47% fatty acids to 5.62%) resulted in a small but statistically significant improvement in the renal cystic disease. Not only do these studies suggest that diet could be a modifiable risk factor for disease, they also highlight the importance of controlling for such in animal and possibly human studies. The diet manipulation in our study was trivial---the two diets are often used interchangeably in mouse colonies. That we can detect a difference highlights the importance of noting that research conducted asynchronously or in different animal facilities within the same institution may not be directly comparable if diet is not standardized. Given that whole organ effects can result from other systemic factors rather than a direct result of loss of Pkd1 function, we sought to determine whether cultured cells lacking Pkd1 had any metabolic abnormalities. As noted in last years annual report, we have developed five different renal epithelial cell line pairs in which one member derived as an isogenic subclone of the other has induced loss of Pkd1 expression. Four of the lines are derived from Pkd1 conditional mice while the fifth pair consists of an original mCCD cell line and a sub-clone in which Pkd1 is silenced by shRNA. Measuring oxygen consumption rate (OCR) with an extracellular flux analyzer, we found lower fatty acid oxidation in all five lines. Surprisingly, we did not find differences in glycolysis as had been recently published by others. Our results suggest that renal epithelial cells lacking Pkd1 have cell autonomous defects in fatty acid oxidation that contribute directly to the in vivo signature of lipid metabolism dysfunction. We published a report describing all of the above findings (transcriptomic, lipidomic, metabolimic, flux analyses and diet studies) this past year. We also have initiated a series of collaborations with NIH investigators to exmaine the lipid properties of our cell lines and very high resolution analyses of cellular structures. Our group also has made steady progress in studying the function of the PKHD1 gene. In our cell culture models, we have found that altering PKHD1 function affects RhoA levels. This effect appears to be mediated by changes in SMURF1 activity. In further characterizing this relationship using cultured cells and a variety of rodent models, we found a novel link between the PKHD1 gene product, FPC (fibrocystin/polyductin), and multiple members of the C2 WWW HECT domain E3 family of ubiquitin ligases. Our results provide a mechanistic explanation for both the cellular effects and important in vivo phenotypic abnormalities observed in mice and humans that result from Pkhd1/PKHD1 mutation. A manuscript describing these findings is under review. We decided to perform additional studies before publishing the Pkhd1flox 67 mouse line. One important new finding is that we have been able to confirm using this mouse line that the Pkhd1 gene product, FPC, undergoes Notch-like processing in tissue as we had predicted based on in vitro cell culture studies. We also have continued to characterize the mouse line that lacks exons 3-67; full characterization of the line is now nearly complete. We have also continued to characterize a mouse line generated in collaboration with the NHLBI mouse core that has a CRISPR-induced deletion within a gene found to be essential for PC2 trafficking in Drosophila. In initial studies, we found normal birth rates and no obvious cystic disease. We have allowed a cohort of mice to age and will soon be exmaining for late onset phenotypes.
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